ΦC31整合酶介导猪基因组定点修饰的探讨  

Site-specific Modification of Pig Genome Mediated by ΦC31 Integrase

作　　者：毕延震[1] 刘西梅[1] 华再东[1] 张立苹[1] 郑新民[1] 肖红卫[1] 

机构地区：[1]动物胚胎工程及分子育种湖北省重点实验室,湖北省农业科学院畜牧兽医研究所,武汉430064

出　　处：《中国生物化学与分子生物学报》2014年第2期187-193,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.31201790);转基因生物新品种培育重大专项(No.2013ZX08010-3;2013ZX08006-003);湖北省自然科学基金重点项目(No.2010CDA120);湖北省农业科学院青年基金重点项目(No.2011NKYJJ13);动物胚胎工程及分子育种湖北省重点实验室开放课题(No.2012ZD121;2013ZD120;121);湖北省农业科技创新中心(No.2011-620-001-003)~~

摘　　要：高效与特异的基因组定点修饰是基因工程动物研究的前沿与难点.链霉菌噬菌体ΦC31整合酶能介导含attB位点的外源基因定点整合于多种真核生物基因组的假attP位点,可维持外源基因的正常结构及高效表达.本文探讨ΦC31整合酶介导外源基因在猪基因组内定点整合的分子基础.构建含attB位点的报告载体pEGFP-N1-attB,与ΦC31整合酶表达载体pCMV-INT共转染猪肾PK15细胞,G418筛选获得单克隆细胞系.实时荧光定量PCR筛选出单拷贝整合的转基因细胞系.TAIL-PCR鉴定出1个猪基因组假attP位点,位于猪1号染色体,watson链,坐标114220087-114220126,命名为pig-attP-1.测序结果显示,pEGFP-N1-attB在attB位点处断开插入到pig-attP-1.用荧光计测定细胞培养基上清EGFP含量发现,该转基因细胞系EGFP的表达水平是本底的50倍(13500 AU vs.280 AU),表明pig-attP-1是利于外源基因高效表达的"友好位点".该研究不仅为实现外源基因在猪基因组内的定点整合提供了新策略,也为创制基因工程猪、建立动物生物反应器等研究注入了新思路.Efficient and targeted genome modification Streptomyces φC31 integrase is capable of integrating is now the hot topic in gene engineering animal. attB-containing plasmid into pseudo attP site in mammalian genomes, resulting in the normal structure and high expression of transgene. This study is to discover the molecular basis of φC31 integrase-mediated site-specific transgene integration in pig genome. A reporter plasmid pEGFP-NI-attB was constructed and co-transfected with φC31 integraseexpressing plasmid pCMV-INT into pig kidney PK15 cells. Cell clones were generated under G418 selection. Quantitative real-time PCR was used to identify cell clones with single-copy transgene integration. One pig pseudo attP site, pig-attP-1 was isolated by TA1L-PCR. It is located in an intergenic region in pig chromosome 1, spanning from 114220087-114220126. Sanger sequencing showed that pEGFP-NI-attP was broken at attB site and joined with pig-attP-1 site. Extracellular EGFP expression was measured by a fluorescence detector, where its expression level was 50 fold higher than that in background fluorescence （13500AU vs. 280AU） , implying that pig-attP-1 site is a safe harbor in favor of transgene expression. This study paves a new way for site-specific transgene integration in pig genome. It also provides a new strategy for creating genetically modified pig and animal bioreactor.

关 键 词：链霉菌噬菌体φC31整合酶 假attP位点 基因工程动物 猪 定点修饰 

分 类 号：Q71[生物学—分子生物学] R73[医药卫生—肿瘤]